The present invention relates to a method for precisely determining the fluorescence in a layer system, such as the eye.
The method can be used for observing the layer-specific fluorescence behavior of endogenous or exogenous fluorophores in organs of biological objects, such as the eye, the skin, the intestine, and the bladder. This invention allows for the control of the diffusion of marked pharmaceuticals through morphological structures and their accumulation at target structures. It can also be used for examining the fluorescence in layered plant structures.
By the invention, a tomographic reconstruction of an object having a structure of fluorescent layers is obtained. The manufacturing control in the production of products with a layered structure is possible, too.
If layer systems are excited to fluorescence, local fluorophores emit in the individual layers in dependence on the specific layer structure, and it is known that the summary decay behavior of the summary fluorescence is observed to evaluate the fluorescence of said fluorophores. In particular, in the human eye, fluorophores are excited in the anatomic layers of the front eye and in the layers of the eye background. The fluorescence both of endogenous fluorophores and of fluorescent markers can be used for diagnostic purposes, such as the assessment of the metabolic state. Thus, the fluorescence analysis of the eye provides information that can be very important for ophthalmologic examinations and for assessing the state of the eye and possible damages and/or for the early detection of diseases of the eye or its components. This information refers to the evaluation of the decay behavior of the substance-specific functional information obtained from the complete object, such as the eye. Moreover, there is still the unresolved problem of gathering information on the layer-specific points of origin of the substance-specific fluorescence.
In addition to this, it would be useful if such measurements and evaluations could be directly performed in the clinical routine of ophthalmological examinations with as little effort and as precise results as possible.
However, the invention is not restricted to applications related to the eye as a layer system.
Generally, the measurements of fluorescence in mixtures of fluorophores are based on the assumption that the substances to be analyzed are located in the same object plane. This assumption applies both for the examination of endogenous fluorophores and of fluorescent markers in cell or tissue cultures.
By means of 2-photon or multi-photon excitation (K. Konig, I. Riemann: High-resolution multi-photon tomography of human skin with subcellular spatial resolution and picosecond time resolution. Journal of Biomedical Optics 8(3), 2003, 432-439), single points of one layer of an object can be excited to fluorescence with a high geometric resolution. In combination with scanning systems (scanner microscopes) the fluorescence of a complete layer can be determined (W. Denk, J. H. Strickler, W. W. Webb: Two-photon laser scanning microscopy, Science 248, 1990, 73-76; B. R. Masters, P. T. C. So, E. Gratton: Multiphoton excitation fluorescence microscopy of in vivo human skin. Ann. N.Y. Acad. Sci. 838, 1998, 58-67). After focusing on further layers, the geometric structure of the fluorescent layers of an object can be principally determined.
To obtain the energy density required for the 2-photon or multi-photon excitation in the focus of the excitation system, optical systems with a high numeric aperture are necessary for high radiation performances. For this purpose immersion objectives of microscopes are useful. They can analyze the fluorescent layers of a microscopic specimen or of the skin up to a layer thickness of about 1 mm in a small field.
For strongly absorbing structures, such as the retinal pigment epithelium, a damage will be already caused if the applied radiation energy is only higher by the factor 3 than the energy used for exciting the fluorescence. The low aperture of the eye determined by the opened iris and the focal length of the eye also excludes the use of 2-photon or multi-photon processes for analyzing the fluorescence of the eye background in the living eye.
The simultaneous determination of the fluorescence of different layers of an object is principally not possible by means of the 2-photon or multi-photon excitation because focusing with high precision can only be realized in one focal plane. Thus, the 2-photon or multi-photon excitation normally does not allow the simultaneous fluorescence measurement of different layers of the eye, for example, of the front and back eye segment.
The examination of the autofluorescence or of the fluorescence of exogenous markers can be carried out at the eye by using a fundus camera or laser scanner ophthalmoscopes. The state-of-the-art is the measurement of static fluorescence, mainly of the eye background (A. von Rückmann, F. W. Fitzke, A. C. Bird: Distribution of fundus autofluorescence with a scanning laser ophthalmoscope, Br. J Ophthalmol 79, 1995, 407-412; F. G. Holz et al.: Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci 42, 2001, 1051-1056). By combining a modified ophthalmoscope with a spectrograph, the fluorescence spectrum of a selected area of the eye background can be calculated (F. C. Delori et al.: In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci 36, 1995, 718-729; D. Schweitzer et al.: Die altersabhängige Makulopathie—Vergleichende Untersuchungen zwischen Patienten, deren Kindern and Augengesunden. (The age-related maculopathy—Comparative examinations of patients, their children and persons with healthy eyes.) Ophthalmologe 97, 2000, 84-90).
In more recent developments, the dynamic fluorescence of the eye is measured after excitation by ps-laser pulses (D. Schweitzer et al.: In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt 9, 2004, 1214-1222; D. Schweitzer et al.: Towards metabolic mapping of the human retina. Microscopy Research and Tech-nique 70, 2007, 410-419). In this method it is assumed for the evaluation of the dynamic (time-resolved) fluorescence of the eye (D. Schweitzer et al.: In vivo measurement of time-resolved autofluorescence at the human fundus, J Biomed Opt 9, 2004, 1214-1222) or of other objects that the fluorescence of all fluorophores has its origin in the same focal plane. A multi-exponential model function according to the equation (1) is, for example, used for the approximation of the summary decay behavior of the fluorescence of the eye as a layer system:
                                          I            ⁡                          (              t              )                                            I            0                          =                                            ∑                              i                =                1                            p                        ⁢                                          α                i                            ·                              ⅇ                                  -                                      t                                          τ                      i                                                                                                    +          b                                    (        1        )            whereinIo: maximum fluorescence intensityI(t): fluorescence at the time tτi: decay time of the component iαi: pre-exponential factor ib: underground intensity.
Since it is assumed in this model function that all fluorophores are localized in only one layer, it is not possible to ascertain the points of origin of the individual fluorescence. Consequently, the time-dependent summary fluorescence of an object that contains several fluorescent layers can be approximately evaluated, but this evaluation is not reliable and inaccurate.